Thermostable glucoamylase

ABSTRACT

The invention relates to an isolated thermostable glucoamylase derived from  Talaromyces emersonii  suitable for starch conversion processes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.09/107,657, filed on Jun. 30, 1998, now abandoned which is a CIP of U.S.application Ser. No. 08/979,673, filed on Nov. 26, 1997, now abandonedand claims priority under 35 U.S.C. 119 of Danish applications 1557/97filed on Dec. 30, 1997 and PA 1998 00925 filed on Jul. 10, 1998 and U.S.Provisionals 60/070,746 filed on Jan. 8, 1998 and 60/094,344 filed onJul. 28, 1998, the contents of which are fully incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to a thermostable glucoamylase suitablefor, e.g., starch conversion, e.g., for producing glucose from starch.The present invention also relates to the use of said thermostableglucoamylase in various processes, in particular in the saccharificationstep in starch convention processes.

BACKGROUND OF THE INVENTION

Glucoamylases (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3) are enzymeswhich catalyze the release of D-glucose from the non-reducing ends ofstarch or related oligo- and polysaccharide molecules.

Glucoamylases are produced by several filamentous fungi and yeasts,including Aspergillus niger and Aspergillus awamori.

Commercially, the glucoamylases are used to convert corn starch which isalready partially hydrolyzed by an α-amylase to glucose. The glucose mayfurther be converted by glucose isomerase to a mixture composed almostequally of glucose and fructose. This mixture, or the mixture furtherenriched with fructose, is the commonly used high fructose corn syrupcommercialized throughout the world. This syrup is the world's largesttonnage product produced by an enzymatic process. The three enzymesinvolved in the conversion of starch to fructose are among the mostimportant industrial enzymes produced.

One of the main problems existing with regard to the commercial use ofglucoamylase in the production of high fructose corn syrup is therelatively low thermal stability of glucoamylases, such as thecommercially available Aspergillus niger glucoamylase (i.e., (sold asAMG by Novo Nordisk A/S). The commercial Aspergillus glucoamylase is notas thermally stable as α-amylase or glucose isomerase and it is mostactive and stable at lower pH's than either α-amylase or glucoseisomerase. Accordingly, it must be used in a separate vessel at a lowertemperature and pH.

U.S. Pat. No. 4,247,637 describes a thermostable glucoamylase having amolecular weight of about 31,000 Da derived from Talaromyces dupontisuitable for saccharifying a liquefied starch solution to a syrup. Theglucoamylase is stated to retain at least about 90% of its initialglucoamylase activity when held at 70° C. for 10 minutes at pH 4.5.

U.S. Pat. No. 4,587,215 discloses a thermostable amyloglucosidasederived from the species Talaromyces thermophilus with a molecularweight of about 45,000 Da. The disclosed amyloglucosidase (orglucoamylase) loses its enzymatic activity in two distinct phases, aninitial period of rapid decay followed by a period of slow decay. At 70°C. (pH=5.0) the half-life for the fast decay is about 18 minutes with nomeasurable loss of activity within an hour in the second phase of decay.Bunni L et al., (1989), Enzyme Microb. Technol., Vol. 11, p. 370-375.concerns production, isolation and partial characterization of anextracellular amylolytic system composed of at least one form ofα-amylase and one form of an α-glucosidase produced by Talaromycesemersonii CBS 814.70. Only the α-amylase is isolated, purified andcharacterized.

BRIEF DISCLOSURE OF THE INVENTION

The present invention is based upon the finding of a novel thermostableglucoamylase suitable for use, e.g., in the saccharification step instarch conversion processes.

The terms “glucoamylase” and “AMG” are used interchangeably below.

The thermal stability of the glucoamylase of the invention is measuredas T_(½) (half-life) using the method described in the “Materials andMethods” section below.

The inventors of the present invention have isolated, purified andcharacterized a thermostable glucoamylase from a strain of Talaromycesemersonii now deposited with the Centraalbureau voor Schimmelculturesunder the number CBS 793.97.

When applied to a protein, the term “isolated” indicates that theprotein is found in a condition other than its native environment. In apreferred form, the isolated protein is substantially free of otherproteins, particularly other homologous proteins (i.e., “homologousimpurities” (see below)).

It is preferred to provide the protein in a greater than 40% pure form,more preferably greater than 60% pure form. Even more preferably it ispreferred to provide the protein in a highly purified form, i.e.,greater than 80% pure, more preferably greater than 95% pure, and evenmore preferably greater than 99% pure, as determined by SDS-PAGE.

The term “isolated enzyme” may alternatively be termed “purifiedenzyme”.

The term “homologous impurities” means any impurity (e.g. anotherpolypeptide than the polypeptide of the invention) which originates fromthe homologous cell, from where the polypeptide of the invention isoriginally obtained.

The isolated glucoamylase has a very high thermal stability incomparison to prior art glucoamylases, such as the Aspergillus nigerglucoamylase (available from Novo Nordisk A/S under the trade name AMG).The T½ (half-life) was determined to be about 120 minutes at 70° C. (pH4.5) as described in Example 2 below. The T½ of the recombinant T.emersonii AMG expressed in yeast was determined to be about 110 minutesas described in Example 12.

Therefore, in the first aspect the present invention relates to anisolated enzyme with glucoamylase activity having a T_(½) (half-life) ofat least 100 minutes in 50 mM NaOAc, 0.2 AGU/ml, pH 4.5, at 70° C.

In the second aspect the invention relates to an enzyme withglucoamylase activity comprising one or more of the partial sequencesshown in SEQ ID Nos. 1-6 or the full length enzyme shown in SEQ ID NO: 7or an enzyme with glucoamylase activity being substantially homologousthereto.

The term “partial sequence” denotes a partial polypeptide sequence whichis comprised in a longer polypeptide sequence, wherein said longerpolypeptide sequence is having the activity of interest.

The invention also relates to the cloned DNA sequence encoding theglucoamylase of the invention.

Further, the invention also relates to a process of converting starch orpartially hydrolyzed starch into a syrup containing, e.g., dextrose,said process including the step of saccharifying starch hydrolyzate inthe presence of a glucoamylase of the invention.

It is an object of the invention to provide a method of saccharifying aliquefied starch solution, wherein an enzymatic saccharification iscarried out using a glucoamylase of the invention.

Furthermore, the invention relates to the use of a glucoamylase of theinvention in a starch conversion process, such as a continuous starchconversion process. In an embodiment of the continuous starch conversionprocess it includes a continuous saccharification step.

The glucoamylase of the invention may also be used in processes forproducing oligosaccharides or specialty syrups.

Finally, the invention relates to an isolated pure culture of themicroorganism Talaromyces emersonii CBS 793.97 or a mutant thereofcapable of producing a glucoamylase of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the SDS-PAGE gel (stained with Coomassie Blue) used fordetermining the molecular weight (M_(w)) of the purified Talaromycesemersonii CBS 793.97 glucoamylase of the present invention.

1: Standard marker,

2: Q Sepharose pool (1. run)

3: S Sepharose pool;

FIG. 2 shows the pH activity profile of Talaromyces emersonii andAspergillus niger glucoamylase (AMG) in 0.5% maltose at 60° C.;

FIG. 3 shows the temperature activity profile of the Talaromycesemersonii CBS 793.97 glucoamylase vs. Aspergillus niger glucoamylase(AMG);

FIG. 4 shows the curve for determining T_(½) (half-life) in 50 mM NaOAc,0.2 AGU/ml, pH 4.5, at 70° C. of Talaromyces emersonii CBS 793.97glucoamylase vs. Aspergillus niger glucoamylase (AMG);

FIG. 5 shows the sequence of the Talaromyces emersonii AMG locus. Thepredicted amino acid sequence is shown below the nucleotide sequence.The four introns are shown in lower case letters. Consensus intronssequences are underlined. Putative signal and pro-peptides are doubleunderlined and dotted underline, respectively;

FIG. 6 shows an alignment/comparison of the amino acid sequences of theA.niger AMG (An_amg-1.pro), A.oryzae AMG Ao_AMG.pro), and Talaromycesemersonii AMG (Tal-AMG.pro). Identical amino acid residues are indicatedby a *. Signal and pro peptides are underlined by a single and a doublelined, respectively;

FIG. 7 shows the Aspergillus expression cassette pCaHj483 used inExample 5;

FIG. 8 shows the Aspergillus expression plasmid, pJal518, for theTalaromyces emersonii AMG gene;

FIG. 9 shows the construction of A.niger disruption plasmid;

FIG. 10 shows the SDS page gel of two transformants, JaL228#5.77 andHowB112#8.10, expressing the Talaromyces emersonii glucoamylase of theinvention. JaL228 and HowB112 are the untransformed parent strains. MW:Promega's Protein Molecular;

FIG. 11 shows the thermal stability of the T. emersonii AMG produced thestrain A. niger HowB112 determined in 50 mM NaOAC, pH 4.5, 70° C., 0.2AGU/ml (T½ determined to 20 minutes);

FIG. 12 compares the thermal stability at 68° C. of the fermentationbroth of T. emersonii AMG expressed in yeast produced in yeast and theA. niger AMG;

FIG. 13 shows the result of the test for determining the thermostabilityof recombinant Talaromyces emersonii AMG produced in yeast at 70° C., pH4.5, 0.2 AGU/ml. T½ was determined to about 110° C.

DETAILED DISCLOSURE OF THE INVENTION

The present invention is based upon the finding of a novel thermostableglucoamylase suitable for use in, e.g., the saccharification step in astarch conversion process.

The inventors of the present invention have isolated, purified andcharacterized a glucoamylase from a strain of Talaromyces emersonii CBS793.97. The glucoamylase turned out to have a very high thermalstability in comparison to prior art glucoamylases.

Accordingly, in a first aspect the present invention relates to anisolated enzyme with glucoamylase activity having a T_(½) (half-life) ofat least 100 minutes, such as between 100 and 140 minutes, in 50 mMNaOAc, 0.2 AGU/ml, pH 4.5, at 70° C.

T½ (half-life) of the isolated Talaromyces emersonii CBS 793.97glucoamylase was determined to be about 120 minutes at 70° C. asdescribed in Example 2 below and to be about 110° C. for the T.emersonii produced in yeast as described in Example 12.

The molecular weight of the isolated glucoamylase was found to be about70 kDa determined by SDS-PAGE. Further, the pI of said enzyme wasdetermined to be below 3.5 using isoelectrical focusing.

The isoelectric point, pI, is defined as the pH value where the enzymemolecule complex (with optionally attached metal or other ions) isneutral, i.e., the sum of electrostatic charges (net electrostaticcharge, NEC) on the complex is equal to zero. In this sum of courseconsideration of the positive or negative nature of the electrostaticcharge must be taken into account.

It is expected that substantially homologous enzymes having the sameadvantageous properties are obtainable from other micro-organisms,especially fungal organisms such as filamentous fungi, in particularfrom another strain of Talaromyces, especially another strains ofTalaromyces emersonii.

The Deposited Micro-organism

An isolate of the filamentous fungus strain, from which the glucoamylaseof the invention has been isolated, has been deposited with theCentraalbureau voor Schimmelcultures, P.O. Box 273, 3740 AG Baarn, theNetherlands, for the purposes of patent procedure on the date indicatedbelow. CBS being an international depository under the Budapest Treatyaffords permanence of the deposit in accordance with rule 9 of saidtreaty.

Deposit date: Jun. 2, 1997

Depositor's ref.: NN049253

CBS designation: CBS 793.97

The isolate of the filamentous fungus Talaromyces emersonii CBS No.793.97 has been deposited under conditions that assure that access tothe isolated fungus will be available during the pendency of this patentapplication to one determined by the commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. § 1.14 and 35 U.S.C §122. The deposit represents a substantially pure culture of the isolatedfungus. The deposit is available as required by foreign patent laws incountries wherein counterparts of the subject application, or itsprogeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

Talaromyces emersonii Glucoamylase Amino Acid Sequence

The inventors have sequenced the thermostable glucoamylase derived fromTalaromyces emersonii CBS 793.97 as will be described further in theExample 3 below. According to the invention the Talaromyces AMG may havea Asp145Asn (or D145N) substitution (using SEQ ID NO: 7 numbering).

Therefore, the invention also relates to an isolated enzyme withglucoamylase activity comprising one or more of the partial sequencesshown in SEQ ID NOS: 1-6 or the full length sequence shown in SEQ ID NO:7 or an enzyme with glucoamylase activity being substantially homologousthereto. SEQ ID NO: 34 shows the full length sequence including thesignal and pre propeptide from amino acid no. 1 to 27.

Homology of the Protein Sequence

The homology between two glucoamylases is determined as the degree ofidentity between the two protein sequences indicating a derivation ofthe first sequence from the second. The homology may suitably bedetermined by means of computer programs known in the art such as gapprovided in the GCG program package (Program Manual for the WisconsinPackage, Version 8, August 1994, Genetics Computer Group, 575 ScienceDrive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D.,(1970), Journal of Molecular Biology, 48, p. 443-453). Using gap withthe following settings for polypeptide sequence comparison: gap creationpenalty of 3.0 and gap extension penalty of 0.1.

According to the invention a “substantially homologous” amino acidsequence exhibits a degree of identity preferably of at least 80%, atleast 90%, more preferably at least 95%, more preferably at least 97%,and most preferably at least 99% with the partial amino acid sequencesshown in SEQ ID NO: 1-6 or SEQ ID NO: 7.

The Cloned Talaromyces emersonii DNA Sequence

The invention also relates to a cloned DNA sequence encoding an enzymeexhibiting glucoamylase activity of the invention, which DNA sequencecomprises:

(a) the glucoamylase encoding part of the DNA sequence shown in SEQ IDNO: 33;

(b) the DNA sequence shown in positions 649-2724 in SEQ ID NO:33 or itscomplementary strand;

(c) an analogue of the DNA sequence defined in (a) or (b) which is atleast 80% homologous with said DNA sequence;

(d) a DNA sequence which hybridizes with a double-stranded DNA probecomprising the sequence shown in 649-2724 in SEQ ID NO: 33 at lowstringency;

(e) a DNA sequence which, because of the degeneracy of the genetic code,does not hybridize with the sequences of (b) or (f), but which codes fora polypeptide having exactly the same amino acid sequence as thepolypeptide encoded by any of these DNA sequences; or

(g) a DNA sequence which is a fragment of the DNA sequences specified in(a), (b), (c), (d), or (e).

The mature part of the AMG of the invention is encoded by the DNAsequence in position 728-2724 of SEQ ID NO: 33. When expressing the AMGof the invention in yeast, e.g., Saccharomyces cerevisiae YNG318, theintrons need to be cut out as described in Example 7.

Homology of DNA Sequences

The DNA sequence homology referred to above is determined as the degreeof identity between two sequences indicating a derivation of the firstsequence from the second. The homology may suitably be determined bymeans of computer programs known in the art, such as GAP provided in theGCG program package (Program Manual for the Wisconsin Package, Version8, August 1994, Genetics Computer Group, 575 Science Drive, Madison,Wis., USA 53711)(Needleman, S. B. and Wunsch, C. D., (1970), Journal ofMolecular Biology, 48, 443-453). Using GAP with the following settingsfor DNA sequence comparison: GAP creation penalty of 5.0 and GAPextension penalty of 0.3, the coding region of the analogous DNAsequences referred to above exhibits a degree of identity preferably ofat least 80%, more preferably at least 90%, more preferably at least95%, more preferably at least 97% with the AMG encoding part of the DNAsequence shown in SEQ ID NO: 33 or the glucoamylase encoding part withor witout introns.

Hybridization:

The hybridization conditions referred to above to define an analogousDNA sequence as defined in d) above which hybridizes to adouble-stranded DNA probe comprising the sequence shown in positions649-2748 in SEQ ID NO: 33 (i.e., the AMG encoding part), under at leastlow stringency conditions, but preferably at medium or high stringencyconditions are as described in detail below.

Suitable experimental conditions for determining hybridization at low,medium, or high stringency between a nucleotide probe and a homologousDNA or RNA sequence involves presoaking of the filter containing the DNAfragments or RNA to hybridize in 5×SSC (Sodium chloride/Sodium citrate,Sambrook et al. 1989) for 10 min, and prehybridization of the filter ina solution of 5×SSC, 5×Denhardt's solution (Sambrook et al. 1989), 0.5%SDS and 100 μg/ml of denatured sonicated salmon sperm DNA (Sambrook etal. 1989), followed by hybridization in the same solution containing aconcentration of 10 ng/ml of a random-primed (Feinberg, A. P. andVogelstein, B. (1983) Anal. Biochem. 132:6-13), ³²P-dCTP-labeled(specific activity >1×10⁹ cpm/μg) probe for 12 hours at about 45° C. Thefilter is then washed twice for 30 minutes in 2×SSC, 0.5% SDS at about55° C. (low stringency), more preferably at about 60° C. (mediumstringency), still more preferably at about 65° C. (medium/highstringency), even more preferably at about 70° C. (high stringency), andeven more preferably at about 75° C. (very high stringency).

Molecules to which the oligonucleotide probe hybridizes under theseconditions are detected using a x-ray film.

Starch Conversion

The present invention provides a method of using the thermostableglucoamylase of the invention for producing glucose and the like fromstarch. Generally, the method includes the steps of partiallyhydrolyzing precursor starch in the presence of α-amylase and thenfurther hydrolyzing the release of D-glucose from the non-reducing endsof the starch or related oligo- and polysaccharide molecules in thepresence of glucoamylase by cleaving α-(1→4) and α-(1→6) glucosidicbonds.

The partial hydrolysis of the precursor starch utilizing α-amylaseprovides an initial breakdown of the starch molecules by hydrolyzinginternal α-(1→4)-linkages. In commercial applications, the initialhydrolysis using α-amylase is run at a temperature of approximately 105°C. A very high starch concentration is processed, usually 30% to 40%solids. The initial hydrolysis is usually carried out for five minutesat this elevated temperature. The partially hydrolyzed starch can thenbe transferred to a second tank and incubated for approximately one hourat a temperature of 85° to 90° C. to derive a dextrose equivalent (D.E.)of 10 to 15.

The step of further hydrolyzing the release of D-glucose from thenon-reducing ends of the starch or related oligo- and polysaccharidesmolecules in the presence of glucoamylase is normally carried out in aseparate tank at a reduced temperature between 30° and 60° C. Preferablythe temperature of the substrate liquid is dropped to between 55° and60° C. The pH of the solution is dropped from 6 to 6.5 to a rangebetween 3 and 5.5. Preferably, the pH of the solution is 4 to 4.5. Theglucoamylase is added to the solution and the reaction is carried outfor 24-72 hours, preferably 36-48 hours.

By using a thermostable glucoamylase of the invention saccharificationprocesses may be carried out at a higher temperature than traditionalbatch saccharification processes. According to the inventionsaccharification may be carried out at temperatures in the range fromabove 60-80° C., preferably 63-75° C. This applies both for traditionalbatch processes (described above) and for continuous saccharificationprocesses.

Actually, continuous saccharification processes including one or moremembrane separation steps, i.e., filtration steps, must be carried outat temperatures of above 60° C. to be able to maintain a reasonably highflux over the membrane. Therefore, a thermostable glucoamylase of theinvention provides the possibility of carrying out large scalecontinuous saccharification processes at a fair price within and periodof time acceptable for industrial saccharification processes. Accordingto the invention the saccharification time may even be shortened.

The activity of a glucoamylase of the invention is generallysubstantially higher at temperatures between 60° C.-80° C. than at thetraditionally used temperature between 30-60° C. Therefore, byincreasing the temperature at which the glucoamylase operates thesaccharification process may be carried out within a shorter period oftime or the process may be carried out using lower enzyme dosage.

As the thermal stability of the glucoamylase of the invention is veryhigh in comparison to, e.g., the commercially available Aspergillusniger glucoamylase (i.e., AMG) a less amount of glucoamylase needs to beadded to replace the glucoamylase being inactivated during thesaccharification process. More glucoamylase is maintained active duringsaccharification process according to the present invention.Furthermore, the risk of microbial contamination is also reduced whencarrying the saccharification process at temperature above 63° C.

By using a glucoamylase with increased specific activity (measured asactivity towards maltose), a lower enzyme dosage may be required in thesaccharification process.

Examples of saccharification processes, wherein the glucoamylase of theinvention may advantageously be used include the processes described inJP 3-224493; JP 1-191693; JP 62-272987; and EP 452,238.

In a further aspect the invention relates to a method of saccharifying aliquefied starch solution, which method comprises an enzymaticsaccharification step using a glucoamylase of the invention.

The glucoamylase of the invention may be used in the present inventiveprocess in combination with an enzyme that hydrolyzes onlyα-(1→6)-glucosidic bonds in molecules with at least four glucosylresidues. Preferably, the glucoamylase of the invention is used incombination with pullulanase or isoamylase. The use of isoamylase andpullulanase for debranching, the molecular properties of the enzymes,and the potential use of the enzymes with glucoamylase is set forth inG.M.A. van Beynum et al., Starch Conversion Technology, Marcel Dekker,New York, 1985, 101-142.

In a further aspect the invention relates to the use of a glucoamylaseof the invention in a starch conversion process.

Further, the glucoamylase of the invention may be used in a continuousstarch conversion process including a continuous saccharification step.

The glucoamylase of the invention may also be used in immobilised form.This is suitable and often used for producing speciality syrups, such asmaltose syrups, and further for the raffinate stream of oligosaccharidesin connection with the production of fructose syrups.

The glucoamylase of the invention may also be used in a process forproducing ethanol for fuel or beverage or may be used in a fermentationprocess for producing organic compounds, such as citric acid, ascorbicacid, lysine, glutamic acid.

MATERIALS AND METHODS Material

Enzymes:

Glucoamylase derived from the deposited filamentous fungus Talaromycesemersonii CBS No. 793.97 hasbeen deposited with the Centraalbureau voorSchimmelcultures, P.O. Box 273, 3740 AG Baarn, the Netherlands, for thepurposes of patent procedure on the date indicated below. CBS being aninternational depository under the Budapest Treaty affords permanence ofthe deposit in accordance with rule 9 of said treaty.

Deposit date: Jun. 2, 1997

Depositor's ref.: NN049253

CBS designation: CBS 793.97

Glucoamylase G1 derived from Aspergillus niger disclosed in Boel et al.(1984), EMBO J. 3 (5), 1097-1102, available from Novo Nordisk and shownin SEQ ID NO: 9.

Strains

JaL228; Construction of this strain is described in WO98/12300 SMO110;Construction of this strain is described in Example 6 Yeast Strain:Saccharomyces cerevisiae YNG318: MATa leu2-D2 ura3-52 his4-539pep4-D1[cir+].

Genes

A. niger G1 glucoamylase gene is shown in SEQ ID NO: 8 T. emersoniiglucoamylase gene with introns is shown in FIG. 5 and SEQ ID NO: 33. Theintrons are shown in FIG. 5.

Plasmids

pJSO026 (S. cerevisiae expression plasmid)(J. S. Okkels, (1996) “AURA3-promoter deletion in a pYES vector increases the expression levelof a fungal lipase in Saccharomyces cerevisiae. Recombinant DNABiotechnology III: The Integration of Biological and EngineeringSciences, vol. 782 of the Annals of the New York Academy of Sciences).More specifically, the expression plasmid pJSO26, is derived from pYES2.0 by replacing the inducible GAL1-promoter of pYES 2.0 with theconstitutively expressed TPI (triose phosphate isomerase)-promoter fromSaccharomyces cerevisiae (Albert and Karwasaki, (1982), J. Mol. ApplGenet., 1, 419-434), and deleting a part of the URA3 promoter.

pJaL497; Construction of this plasmid is described in Example 5

pJaL507; Construction of this plasmid is described in Example 5

pJaL510; Construction of this plasmid is described in Example 5

pJaL511; Construction of this plasmid is described in Example 5

pJaL518; Construction of this plasmid is described in Example 6

pCaHj483; Construction of this plasmid is described in Example 6

pJRoy10; Construction of this plasmid is described in Example 6

pJRoy17; Construction of this plasmid is described in Example 6

pSMO127; Construction of this plasmid is described in Example 6

PCR™II; Available from Invitrogen Corporation, San Diego, Calif., USA.

Equipment:

Automatic DNA Sequencer (Applied Biosystems Model 377)

Media:

SC-ura medium: Yeast Nitrogen w/o ami 7.5 g Bernsteinsaüre (Ravsyre)11.3 g NaOH 6.8 g Casaminoacid w/o vit 5.6 g Tryptophan 0.1 g Dest.water ad 1000 ml

Autoclaved for 20 minutes at 121° C. From a sterile stock solution of 5%Threonin 4 ml is added to a volume of 900 ml together with 100 ml of asterile 20% glucose.

YPD medium:

Yeast extract 10 g Peptone 20 g Dest. water ad 1000 ml

Autoclaved for 20 minutes at 121° C. 100 ml of a sterile 20% glucose isadded to 900 ml.

Methods

Determination of AGU Activity

One Novo Amyloglucosidase Unit (AGU) is defined as the amount of enzymewhich hydrolyzes 1 micromole maltose per minute under the followingstandard conditions:

Substrate maltose Temperature 25° C. pH 4.3 (acetate buffer) Reactiontime 30 minutes

A detailed description of the analytical method (AF22) is available onrequest.

Determination of PUN Activity

PUN is defined as the amount of enzyme which hydrolyzes pullulan (0.2%pullulan, 40° C., pH 5.0), liberating reducing carbohydrate with areducing power equivalent to 1 micro-mol glucose pr. minute.

Determination of AFAU Activity

The activity is determined in AFAU calculated as the reduction in starchconcentration at pH 2.5, 40° C., 0.17 g/l starch and determined by aniodine-starch reaction.

Thermal Stability I (T½ (half-life) Determination of AMG

The thermal stability of glucoamylase (determined as T½ (half-life)) istested using the following method: 950 microliter 50 mM sodium acetatebuffer (pH 4.5) (NaOAc) is incubated for 5 minutes at 70° C. 50microliter enzyme in buffer (4 AGU/ml) is added. 2×40 microliter samplesare taken at fixed periods between 0 and 360 minutes and chilled on ice.After chilling the samples the residual enzyme activity is measuredusing the AGU determination assay (described above).

The activity (AGU/ml) measured before incubation (0 minutes) is used asreference (100%). T_(½) is the period of time until which the percentrelative activity is decreased to 50%.

Determination of Thermal Stability II

1600 microliter of a supernatant and 400 microliter of 0.5M NaAC pH 4.5is mixed.

7 eppendorph tubes each containing 250 microliter of the mixture areincubated in a Perkin Elmer thermocycler at 68° C. or 70° C. for 0, 5,10, 20, 30, 45 and 60 minutes.

100 microliter from each mixture is mixed with 100 microliter of 5 mMCNPG3 (2-chloro-4-Nitrophenyl-Alpha-Maltotrioside from genzyme) inmicrotiterwells. After incubation for 30 minutes at 37° C. theabsorbance is measured at 405 nm.

Determination of Specific Activity of a Glucoamylase

750 microL substrate is incubated 5 minutes at selected temperatures,such as 37° C., 60° C. or 70° C.

50 microL enzyme diluted in sodium acetate is added, and the activitywas determined using the AGU standard method described above. Thekinetic parameters: Kcat and Km are measured at 45° C. by adding 50microL enzyme diluted in sodium acetate to preheated 750 microLsubstrate. Aliquots of 100 microL are removed after 0, 3, 6, 9 and 12minutes and transferred to 100 microL 0.4M Sodium hydroxide to stop thereaction. A blank is included.

20 microL is transferred to a Micro titre plates and 200 microLGOD-Perid solution is added. Absorbance is measured at 650 nm after 30minutes incubation at room temperature. Glucose is used as standard, andthe specific activity is calculated as k_(cat) (sec.⁻¹)

Transformation of Aspergillus oryzae (general procedure)

100 ml of YPD (Sherman et al., (1981), Methods in Yeast Genetics, ColdSpring Harbor Laboratory) is inoculated with spores of A. oryzae andincubated with shaking for about 24 hours. The mycelium is harvested byfiltration through miracloth and washed with 200 ml of 0.6 M MgSO₄. Themycelium is suspended in 15 ml of 1.2 M MgSO₄, 10 mM NaH₂PO₄, pH 5.8.The suspension is cooled on ice and 1 ml of buffer containing 120 mg ofNovozym™ 234 is added. After 5 min., 1 ml of 12 mg/ml BSA (Sigma typeH25) is added and incubation with gentle agitation continued for 1.5-2.5hours at 37C until a large number of protoplasts is visible in a sampleinspected under the microscope.

The suspension is filtered through miracloth, the filtrate transferredto a sterile tube and overlayed with 5 ml of 0.6 M sorbitol, 100 mMTris-HCl, pH 7.0. Centrifugation is performed for 15 min. at 1000 g andthe protoplasts are collected from the top of the MgSO₄ cushion. 2volumes of STC (1.2 M sorbitol, 10 mM Tris-HCl, pH 7.5, 10 mM CaCl₂) areadded to the protoplast suspension and the mixture is centrifugated for5 min. at 1000 g. The protoplast pellet is resuspended in 3 ml of STCand repelleted. This is repeated. Finally, the protoplasts areresuspended in 0.2-1 ml of STC.

100 μl of protoplast suspension are mixed with 5-25 μg of p3SR2 (an A.nidulans amdS gene carrying plasmid described in Hynes et al., Mol. andCel. Biol., Vol. 3, No. 8, 1430-1439, August 1983) in 10 μl of STC. Themixture is left at room temperature for 25 min. 0.2 ml of 60% PEG 4000(BDH 29576), 10 mM CaCl₂ and 10 mM Tris-HCl, pH 7.5 is added andcarefully mixed (twice) and finally 0.85 ml of the same solution areadded and carefully mixed. The mixture is left at room temperature for25 min., spun at 2.500 g for 15 min. and the pellet is resuspended in 2ml of 1.2M sorbitol. After one more sedimentation the protoplasts arespread on minimal plates (Cove, (1966), Biochem. Biophys. Acta 113,51-56) containing 1.0 M sucrose, pH 7.0, 10 mM acetamide as nitrogensource and 20 mM CsCl to inhibit background growth. After incubation for4-7 days at 37C spores are picked, suspended in sterile water and spreadfor single colonies. This procedure is repeated and spores of a singlecolony after the second re-isolation are stored as a definedtransformant.

Fed Batch Fermentation

Fed batch fermentation is performed in a medium comprising maltodextrinas a carbon source, urea as a nitrogen source and yeast extract. The fedbatch fermentation is performed by inoculating a shake flask culture offungal host cells in question into a medium comprising 3.5% of thecarbon source and 0.5% of the nitrogen source. After 24 hours ofcultivation at pH 5.0 and 34° C. the continuous supply of additionalcarbon and nitrogen sources are initiated. The carbon source is kept asthe limiting factor and it is secured that oxygen is present in excess.The fed batch cultivation is continued for 4 days, after which theenzymes can be recovered by centrifugation, ultrafiltration, clearfiltration and germ filtration. Further purification may be done byanionexchange chromatographic methods known in the art.

Transformation of Saccharomyces cerevisiae YNG318

The DNA fragments and the opened vectors are mixed and transformed intothe yeast Saccharomyces cerevisiae YNG318 by standard methods.

EXAMPLES Example 1

Purification

3500 ml T. emersonii culture broth from wild-type fermentation with 0.05AGU/ml was centrifuged at 9000 rpm followed by vacuum filtration throughfilter paper and finally a blank filtration. The following procedure wasthen used to purify the enzyme:

Phenyl Sepharose (250 ml): 1,3 M AMS/10 mM Tris/2 mM CaCl₂, pH 7;elution with 10 mM Tris/2 mM CaCl₂, pH 7.

Dialysis: 20 mM NaAc, 2 mM CaCl₂, pH 5.

Q Sepharose (100 ml): 20 mM NaAc, 2 mM CaCl₂, pH 5; elution with alinear gradient from 0-0.4 M NaCl over 10 column volumes.

Dialysis: 20 mM NaAc, 2 mM CaCl₂, pH 5.

Colour removal: 0.5% coal in 10 minutes.

Q Sepharose (20 ml): 20 mM NaAc, 2 mM CaCl₂, pH 4.5; elution with alinear gradient from 0-0.4 M NaCl over 10 column volumes.

Dialysis: 20 mM NaAc, 2 mM CaCl₂, pH 5.

S Sepharose (1 ml): 5 mM citric acid, pH 2.9; elution with a lineargradient from 0-0.3 M NaCl over 10 column volume.

A purity of the enzyme of more than 90% was obtained after the SSepharose step.

Example 2

Characterisation of the Talaromyces emersonii Glucoamylase

The purified Talaromyces emersonii CBS 793.97 glucoamylase was used forcharacterisation.

Molecular Weight (M_(w))

The molecular weight was determined by SDS-PAGE to around 70 kDa asshown in FIG. 1.

pI

The pI was determined to lie below 3.5 by isoelectrical focusing(Amploline PAG, pH 3.5-9.5 from Pharmacia).

pH Profile

The pH-activity dependency of the Talaromyces emersonii glucoamylase wasdetermined and compared with profile of Aspergillus niger glucoamylase.

The pH activity profile was determined using 0.5% maltose as substratein 0.1 M sodium acetate at 60° C. The pH was measured in duple samplescomprising 0.1-1 AGU/ml. The result of the test is shown in FIG. 2.

Temperature Profile

The temperature-activity dependency of the Talaromyces emersoniiglucoamylase of the invention was determined and compared with theprofile of Aspergillus niger glucoamylase. 200 μl 0.5% maltose, pH 4.3was incubated at 37, 50, 60, 70, 75, 80 and 90° C. and the reaction wasstarted by adding 10 μl enzyme (0.25 AGU/ml); reaction time was 10minutes. The result of the test is shown in FIG. 3.

Temperature Stability—T½ (half-life)

The thermal stability of the Talaromyces emersonii glucoamylase wasdetermined and compared with the thermal stability of Aspergillus nigerglucoamylase. The method used is described above in the “Material andMethods” section as “Thermal Stability I (T½ (half-life) determinationof AMG”.

The T½ of the Talaromyces emersonii glucoamylase was determined to about120 minutes at 70° C. The T½ of the Aspergillus niger glucoamylase wasdetermined to 7 minutes under the same conditions (See FIG. 4).

Specific Activity

The extension coefficient was determined to: ε=2.44 ml/mg*cm on basis ofabsorbency at 280 nm and protein concentration. The specific activitytowards maltose at 37° C. was then calculated to 7.3 AGU/mg. Purity ofthe sample was approximately 90% and a corrected specific activity istherefore 8.0 AGU/mg. Following specific activities were measured:

Specific activity (AGU/mg) AMG 37° C. 60° C. 70° C. T. emersonii* 8.0 2127 A. niger 2.0 6.6 8.0 *Estimated for pure enzyme.

Example 3

Sequencing of the N-terminal of T. emersonii Glucoamylase

The N-terminal amino acid sequence of T. emersonii glucoamylase wasdetermined following SDS-PAGE and electroblotting onto a PVDF-membrane.Peptides were derived from reduced and S-carboxymethylated glucoamylaseby cleaving with a lysyl-specific protease. The resulting peptides werefractionated and re-purified using RP-HPLC before subjected toN-terminal sequence determination.

N-terminal sequence (SEQ ID NO: 1):

Ala Asn Gly Ser Leu Asp Ser Phe Leu Ala Thr Glu Xaa Pro Ile Ala Leu GlnGly Val Leu Asn Asn Ile Gly

Peptide 1 (SEQ ID NO: 2):

Val Gln Thr Ile Ser Asn Pro Ser Gly Asp Leu Ser Thr Gly Gly Leu Gly GluPro Lys

Peptide 2 (SEQ ID NO: 3):

Xaa Asn Val Asn Glu Thr Ala Phe Thr Gly Pro Xaa Gly Arg Pro Gln Arg AspGly Pro Ala Leu

Peptide 3 (SEQ ID NO: 4):

Asp Val Asn Ser Ile Leu Gly Ser Ile His Thr Phe Asp Pro Ala Gly Gly CysAsp Asp Ser Thr Phe Gln Pro Cys Ser Ala Arg Ala Leu Ala Asn His Lys

Peptide 4 (SEQ ID NO: 5):

Thr Xaa Ala Ala Ala Glu Gln Leu Tyr Asp Ala Ile Tyr Gln Trp Lys

Peptide 5 (SEQ ID NO: 6):

Ala Gln Thr Asp Gly Thr Ile Val Trp Glu Asp Asp Pro Asn Arg Ser Tyr ThrVal Pro Ala Tyr Cys Gly Gln Thr Thr Ala Ile Leu Asp Asp Ser Trp Gln

Xaa denoted a residue that could not be assigned.

Example 4

The Full Length T. emersonii Glucoamylase

The full length T. emersonii glucoamylase amino acid sequence shown inSEQ ID NO: 7 was identified using standard methods.

Example 5

Cloning and Sequencing of the Talaromyces emersonii Glucoamylase Gene

PCR cloning parts of the Talaromyces emersonii AMG gene

For cloning of the Talaromyces emersonii AMG gene degenerated primersshown in table 1 was designed for PCR amplification of part of the AMGgene.

TABLE 1 Primer no: Sequence Comments     V  L  N  N  I  G N-Terminal102434 (SEQ ID NO:10) 5′ -GTNTTRAAYAAYATHGG 102435 (SEQ ID NO:11)5′ -GTNCTNAAYAAYATHGG 5′ primers  D  L  W  E  E  V Active site 117360(SEQ ID NO:12) CTRGANACCCTYCTYCA-5′ consensus 3′ primers 117361 (SEQ IDNO:13) CTRAAYACCCTYCTYCA-5′  W  E  D  D  P  N C-Terminal 127420 (SEQ IDNO:14) ACCCTYCTRCTRGGNTT-5′ 3′ primers

Genomic DNA from Talaromyces emersonii was prepared from protoplastsmade by standard procedures [cf.e.g., Christensen et. al. Biotechnology1989 6 1419-1422] and was used as template in the PCR reaction.Amplification reaction were performed in 100 μl volumes containing 2.5units Taq-polymerase, 100 ng of A.oryzae genomic DNA, 50 mM KCl, 10 mMTris-HCl pH 8.0,1.5 mM MgCl₂, 250 nM of each dNTP, and 100 pM of each ofthe following primers sets: 102434/117360, 102434/117361, 102435/117360,102434/117361, 102434/127420, and 102434/127420.

Amplification was carried out in a Perkin-Elmer Cetus DNA Termal 480,and consisted of one cycle of 3 minutes at 94° C., followed by 30 cyclesof 1 minutes at 94° C., 30 seconds at 40° C., and 1 minutes at 72° C.Only the PCR reaction 102434/117360 gave products. Four bands wasdetected with the following sizes 1400, 800, 650, and 525 bp. All fourbands were purified and cloned into the vector pCR®2.1 (Invitrogen®).Sequencing of a few clone from each band and sequence comparisons to theA.niger AMG, releaved that a clone from the 650 bp band encodes for theN-terminal part of the Talaromyces emersonii AMG. This clone wasdesignated pJaL497.

To obtained more of the gene a specific primer (123036:5′-GTGAGCCCAAGTTCAATGTG-3′ (SEQ ID NO:15) was made out from the sequenceof clone pJaL497. The primer set 123036/127420 was used for PCR onTalaromyces genomic DNA and a single fragment on 1500 bp was obtained.The PCR fragment was clone into the vector pCR®2.1 and sequenced. Bysequencing the clone was confirmed to encoded the C-terminal part of theTalaromyces emersonii AMG. The clone was designated pJaL507.

Genomic Restriction Mapping and Cloning of a Genomic Clone(s)

Taken together the two clones pJaL497 and pJaL507 covered about 95% ofthe AMG gene. In order to clone the missing part of the AMG gene agenomic restriction map was constructed by using the two PCR fragment asprobes to a Southern blot of Talaromyces emersonii genomic DNA digestedwith single or a combination of a number of restriction enzymes. Thisshows that the Talaromyces emersonii AMG gene is located on two EcoRIfragment on about 5.6 kb and 6.3 kb, respectively.

Talaromyces emersonii genomic DNA was digested with EcoRI and fragmentswith the size between 4-7 kb was purified and used for construction of apartially genome library in Lambda ZAP II as described by themanufactory instruction(Stratagene). The library was first screenedusing the 0.7 kb EcoRI fragment from pJaL497 (encoding the N-terminalhalf of the AMG gene) as probe to get the start of the AMG gene. Oneclone was obtained and designated pJaL511. In a second screening of thelibrary using a 0.75 kb EcoRV fragment from pJaL507 (encoding theC-terminal half of the AMG gene) as probe in order to get the C-terminalend of the AMG gene. One clone was obtained and designated pJaL510.

Sequence Analysis of the Talaromyces emersonii AMG Gene

The AMG gene sequence was obtained by sequencing on the plasmids:pJaL497, pJaL507, pJaL510, and pJaL511 and on subclones hereof with thestandard reverse and forward primers for pUC. Remaining gabs were closedby using specific oligonucleotide as primers.

Potential introns were found by comparing the sequence with consensussequences for introns in Aspergillus and with the A.niger AMG sequence.The Talaromyces emersonii AMG nucleotide sequence has an open readingframe encoding a protein on 618 amino acid, interrupted by four intronsof 57 bp, 55 bp, 48 bp, and 59 bp, respectively. The nucleotide sequence(with introns) and deduced amino acid sequence is shown in FIG. 5. TheDNA sequence (with introns) is also shown in SEQ ID NO: 33 and theTalaromyces emersonii AMG sequence (with signal sequence from 1 to 27)is shown in SEQ ID NO: 34. Comparison of the deduced amino acid sequencewith the A.oryzae AMG and A.niger AMG shows an identity of 60.1% and60.5%, respectively. Alignment of the amino acid sequences shown in FIG.6 shows that the Talaromyces AMG has a very short hinge between thecatalytic domain and the starch binding domain, which is also seen forthe A.oryzae AMG.

Example 6

Construction of the Aspergillus Vector pCaHj483

Construction of pCaHj483 is depicted in FIG. 7. Said plasmid is buildfrom the following fragments:

a) The vector pToC65 (WO 91/17243) cut with EcoRI and XbaI.

b) A 2.7 kb XbaI fragment from A. nidulans carrying the amdS gene (C. M.Corrick et al., Gene 53, (1987), 63-71). The amdS gene is used as aselective marker in fungal transformations. The amdS gene has beenmodified so that the BamHI site normally present in the gene isdestroyed. This has been done by introducing a silent point mutationusing the primer:

5′-AGAAATCGGGTATCCTTTCAG-3′ (SEQ ID NO:16)

c) A 0.6 kb EcoRI/BamHI fragment carrying the A. niger NA2 promoterfused to a 60 bp DNA fragment of the sequence encoding the 5′untranslated end of the mRNA of the A. nidulans tpi gene. The NA2promoter was isolated from the plasmid pNA2 (described in WO 89/01969)and fused to the 60 bp tpi sequence by PCR. The primer encoding the 60bp tpi sequence had the following sequence:

5′-GCTCCTCATGGTGGATCCCCAGTTGTGTATATAGAGGATTGAGGAAGGAAGAGAAGTGTGGATAGAGGTAAATTGAGTTGGAAACTCCAAGCATGGC ATCCTTGC - 3′ (SEQ IDNO: 17)

d) A 675 bp XbaI fragment carrying the A. niger glucoamylasetranscription terminator. The fragment was isolated from the plasmidpICAMG/Term (described in EP 0238 023).

The BamHI site of fragment c was connected to the XbaI site in front ofthe transcription terminator on fragment d via the pIC19R linker (BamHIto XbaI)

Construction of a AMG Expression Plasmid, pJaL518

The coding region of the Talaromyces emersonii AMG gene was amplified byPCR, using the following two oligonucleotides primers: 139746:

5′-GACAGATCTCCACCATGGCGTCCCTCGTTG 3′ (SEQ ID NO:18); and primer 139747:

5′-GACCTCGAGTCACTGCCAACTATCGTC 3′ (SEQ ID NO:19). The underlined regionsindicate sequences present in the Talaromyces emersonii AMG gene. Tofacilitate cloning a restriction enzyme site was inserted into the 5′end of each primer; primer 139746 contains a BglII site and primer139747 contains a XhoI site. Talaromyces emersonii genomic DNA was usedas template in the PCR reaction. The reaction was performed in a volumeof 100 μl containing 2.5 units Taq polymerase, 100 ng of pSO2, 250 nM ofeach dNTP, and 10 pmol of each of the two primers described above in areaction buffer of 50 mM KCl, 10 mM Tris-HCl pH 8.0, 1.5 mM MgCl₂.

Amplification was carried out in a Perkin-Elmer Cetus DNA Termal 480,and consisted of one cycle of 3 minutes at 94° C., followed by 25 cyclesof 1 minute at 94° C., 30 seconds at 55° C., and 1 minute at 72° C. ThePCR reaction produced a single DNA fragment of 2099 bp in length. Thisfragment was digested with BglII and XhoI and isolated by gelelectrophoresis, purified, and cloned into pCaHj483 digested with BamHIand XhoI, resulting in a plasmid which was designated pJaL518. Thus, theconstruction of the plasmid pJal518 resulted in a fungal expressionplasmid for the Talaromyces emersonii AMG gene (FIG. 8).

Construction of the Aspergillus niger Strain, SMO110

1. Cloning of A.niger pyrG Gene

A library of A.niger BO-1 was created in EMBL4 as described by themanufactory instructions. The library was screened with a DIG labelledoligonucleotides (PyrG: 5′-CCCTCACCAGGGGAATGCTGCAGTTGATG-3′ (SEQ IDNO:20) which was designed from the published Aspergillus niger sequence(Wilson et al. Nucleic Acids Res. 16, (1988), 2339-2339). A positiveEMBL4 clone which hybridized to the DIG probe was isolated from the BO-1library, and a 3.9 kb Xbal fragment containing the pyrG gene wassubcloned from the EMBL4 clone and clone into pUC118 to create pJRoy10.

2. Cloning of the A.niger Glucoamylase (AMG) Gene

The above A.niger BO-1 library was screened with a DIG labelled PCRfragment generated by amplification on A.niger genomic DNA with thefollowing oligonucleotides, 950847:

5′-CGCCATTCTCGGCGACTT-3′ (SEQ ID NO:21), and oligonucleotide 951216:

5′-CGCCGCGGTATTCTGCAG-3′ (SEQ ID NO:22), which was designed from thepublished Aspergillus niger sequence (Boel et al., EMBO J. 3, (1984),1581-1585). A positive EMBL4 clone which hybridized to the DIG probe wasisolated from the BO-1 library, and a 4.0 kb SpeI fragment containingthe AMG gene was subcloned from the EMBL4 clone and clone intopBluescriptSK+ generating plasmid pJRoy17a.

3. Construction of the A. niger AMG Disruption Cassette

A 2.3 kb SpeI-XhoI fragment containing pyrG was gel isolated frompJRoy10 and the restricted ends filled in with Klenow polymerase. Thefragment was inserted into the BglII site of pJRoy17 which cuts withinthe AMG gene creating plasmid pSMO127 (FIG. 9). Between the two SpeIsites of pSMO127a is contained the 2.3 kb pyrG gene flanked by 2.2 kband 2.3 kb 5′ and 3′ AMG, respectively.

4. Construction of a A. niger Strain Disrupted for AMG, SMO110

A.niger JRoyP3 is a spontaneously pyrG mutant of A.niger BO-1, which wasselected for the growth on a plate containing 5′-fluoro-orotic acid(5′-FOA). The pyrG gene encodes orotidine 5′-phosphate carboxylase andits deficient mutant can be characterized as uridine auxotroph. Theidentity of pyrG mutant was confirmed by the complementation of thegrowth on a minimal medium with A.nidulans pyrG gene.

Twenty micrograms of the plasmid pSMO127 was digested with SpeI. The DNAwas resolved on an 0.8% agarose gel and the 6 kb consisting of thelinear disruption cassette was gel isolated. The linear DNA wastransformed into strain JRoyP3. Genomic DNA was prepared from 200transformants which was then digested with SpeI. The gel-resolved DNAwas transferred to a hybond nylon filter, and hybridized to anon-radioactive DIG probe consisting of the AMG open reading frame. Agene replacement of the disruption cassette into the AMG locus wouldresult in an increase of the wild type 4 kb AMG band to 6.3 kb, anincrease due to the 2.3 kb pyrG gene. One transformant #110 with theabove characteristics was selected for further analysis.

The transformant #110 were grown in 25 ml shake flasks containing YPMmedia. Strains BO-1 and parent strain JRoyP3 were grown as AMG producingcontrols. After 3 days, 30 μl of clear supernatants were run on a 8-16%SDS PAGE Novex gel. No AMG band was seen in transformant #110, whilelarge bands of AMG were produced in the positive control strain BO-1 andparent strain JRoyP3. Transformant #110 was named SMO110.

Expression of Talaromyces emersonii AMG in Aspergillus oryzae andAspergillus niger

The strains JaL228 and SMO110 was transformed with pJaL518 as describedby Christensen et al.; Biotechnology 1988 6 1419-1422. Typically, A.oryzae mycelia was grown in a rich nutrient broth. The mycelia wereseparated from the broth by filtration. The enzyme preparation Novozyme®(Novo Nordisk) was added to the mycelia in osmotically stabilizingbuffer such as 1.2 M MgSO₄ buffered to pH 5.0 with sodium phosphate. Thesuspension was incubated for 60 minutes at 37° C. with agitation. Theprotoplast was filtered through mira-cloth to remove mycelial debris.The protoplast was harvested and washed twice with STC (1.2 M sorbitol,10 mM CaCl₂, 10 mM Tris-HCl pH 7.5). The protoplast was finallyresuspended in 200-1000 μl STC.

For transformation 5 μg DNA was added to 100 μl protoplast suspensionand then 200 μl PEG solution (60% PEG 4000, 10 mM CaCl₂, 10 mM Tris-HClpH 7.5) was added and the mixture was incubated for 20 minutes at roomtemperature. The protoplast were harvested and washed twice with 1.2 Msorbitol. The protoplast was finally resuspended 200 μl 1.2 M sorbitol,plated on selective plates (minimal medium+10 g/l Bacto-Agar (Difco),and incubated at 37° C. After 3-4 days of growth at 37° C., stabletransformants appear as vigorously growing and sporulating colonies.Transformants was spore isolated twice.

Transformants was grown in shake flask for 4 days at 30° C. in 100 mlYPM medium (2 g/l yeast extract, 2 g/l peptone, and 2% maltose).Supernatants were tested for AMG activity as described and analyzed onSDS page gel (FIG. 10).

Example 7

Removal of the Four Introns from the Talaromyces emersonii AMG DNASequence for Expression in Yeast.

For each exon a PCR reaction was made with primers containing overlap tothe next exon. Tal 1 and Tal 4 contain an overlap with the yeast vectorpJSO026. Exon 1: Tal 1 was used as the 5′ primer and Tal 5 as the 3′primer and the genomic sequence coding for AMG was used as the template.Exon 2: Tal 6 was used as the 5′ primer and Tal 7 was used as the 3′primer and the genomic sequence coding for AMC was used as the template.Exon 3: Tal 8 was used as the 5′ primer and Tal 9 was used as the 3′primer and the genomic sequence coding for AMG was used as the template.Exon 4: Tal 10 was used as the 5′ primer and Tal 11 was used as the 3′primer and the genomic sequence coding for AMG was used as the template.Exon 5: Tal 12 was used as the 5′ primer and Tal 4 was used as the 3′primer and the genomic sequence coding for AMG was used as the template.

A final PCR reaction was performed to combine the 5 exons to a sequencecontaining the complete coding sequence. In this PCR reaction the 5fragments from the first PCR reactions were used as template and Tal 1was used as the 5′ primer and Tal4 was used as the 3′ primer.

This final PCR fragment containing the coding region was used in an invivo recombination in yeast together with pJSO026 cut with therestriction enzymes SmaI(or BamHI) and XbaI (to remove the coding regionand at the same time create an overlap of about 20 bp in each end tomake a recombination event possible).

Tal 1: 5′-CAA TAT AAA CGA CGG TAC CCG GGA GAT CTC CAC CATG GCG TCC CTCGTT G-3′ (SEQ ID NO:23); Tal 4: 5′-CTA ATT ACA TCA TGC GGC CCT CTA GATCAC TGC CAA CTA TCG TC-3′ (SEQ ID NO:24); Tal 5: 5′-AAT TTG GGT CGC TCCTGC TCG-3′ (SEQ ID NO:25); Tal 6: 5′-CGA GCA GGA GCG ACC CAA ATT ATT TCTACT CCT GGA CAC G-3′ (SEQ ID NO:26); Tal 7: 5′-GAT GAG ATA GTT CGC ATACG-3′ (SEQ ID NO:27); Tal 8: 5′-CGT ATG CGA ACT ATC TCA TCG ACA ACG GCGAGG CTT CGA CTG C-3′ (SEQ ID NO:28); Tal 9: 5′-CGA AGG TGG ATG AGT TCCAG-3′ (SEQ ID NO:29); Tal 10: 5′-CTG GAA CTC ATC CAC CTT CGA CCT CTG GGAAGA AGT AGA AGG-3′ (SEQ ID NO:30) Tal 11: 5′-GAC AAT ACT CAG ATA TCCATC-3′ (SEQ ID NO:31) Tal 12: 5′-GAT GGA TAT CTG AGT ATT GTC GAG AAA TATACT CCC TCA GAC G-3′ (SEQ ID NO:32)

Example 8

Expression of Talaromyces emersonii Glucoamylase in Yeast

To express Talaromyces emersonii AMG in the yeast Saccharomycescerevisiae YNG318 the yeast expression vector pJSO26 was constructed asdescribed in the “Material and Methods” section above.

PJSO26 comprising the DNA sequence encoding the Talaromyces AMG wastransformed into the yeast by standard methods (cf. Sambrooks et al.,(1989), Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold SpringHarbor)

The yeast cells were grown at 30° C. for 3 days in Sc-ura mediumfollowed by growth for 3 days in YPD. The culture was then centrifugedand the supernatant was used for the thermostability assay described inthe “Materials and Method” section.

Thermal Stability of the Talaromyces AMG Expressed in Yeast at 68° C.

The fermentation broth of the Talaromyces emersonii AMG expressed inyeast (Saccharomyces cerevisiae YNG318) was used for determination ofthe thermal stability at 68° C. using the method described above under“Determination of thermal stability II”. The result of the test is shownin FIG. 12.

Example 9

Purification of Recombinant Talaromyces AMG Produced using A. nigerHowB112

200 ml culture broth from fermentation of A. niger HowB112 harboring theTalaromyces emersonii gene was centrifuged at 9000 rpm and dialyzedagainst 20 mM NaOac, pH 5 over night. The solution was then applied on aS Sepharose column (200 ml) previously equilibrated in 20 mM NaOAc, pH5. The glucoamylase was collected in the effluent, and applied on a QSepharose column (50 ml) previously equilibrated in 20 mM NaOAC, pH 4.5.Unbound material was washed of the column and the glucoamylase waseluted using a linear gradient from 0-0.3 M NaCl in 20 mM NaOAc over 10column volumes. Purity of the glucoamylase fraction was checked bySDS-PAGE and only one single band was seen. The molecular weight wasagain found to about 70 kdal as seen for the wild type glucoamylase. Thespecific activity towards maltose was measured and a specific activityof 8.0 AGU/mg (37° C.) and 21.0 AGU/mg (60° C.) were found which is inaccordance the data on the wild type enzyme.

Example 10

Kinetic Parameters

Kinetic Parameters for Hydrolysis of Maltose and Isomaltose byAspergillus niger AMG and the recombinant Talaromyces emersonii AMGexpressed in A. niger.

k_(cat) (s⁻¹)^(a) K_(m) (mM) k_(cat)/K_(m) (s⁻¹mM⁻¹) Maltose Talaromycesemersonii 30.6 3.8 8.1 Aspergillus niger 10.7 1.2 8.8 IsomaltoseTalaromyces emersonii 2.70 53.6 0.050 Aspergillus niger 0.41 19.8 0.021^(a)At 45° C. uusing 0.05M NaOAc, pH 4.5

Example 11

Saccharification Performance of Recombinant Talaromyces emersonii AMGProduced in A. niger

The saccharification performance of the Talaromyces emersoniiglucoamylase was tested at different temperatures with and without theaddition of acid α-amylase and pullulanase. Saccharification was rununder the following conditions: Substrate: 10 DE Maltodextrin, approx.30% DS (w/w) Temperatures: 60, 65, or 70° C. Initial pH: 4.5 Enzymedosage: Recombinant Talaromyces emersonii glucoamylase produced in A.niger: 0.24 or 0.32 AGU/g DS Acid α-amylase derived from A. niger: 0.020AFAU/g DS Pullulanase derived from Bacillus: 0.03 PUN/g DS

When used alone Talaromyces AMG was dosed at the high dosage (0.32 AGU/gDS), otherwise at the low dosage, i.e., 0.24 AGU/g DS.

Saccharification

The substrate for saccharificationg was made by dissolving maltodextrin(prepared from common corn) in boiling Milli-Q water and adjusting thedry substance to approximately 30% (w/w). pH was adjusted to 4.5(measured at 60° C.). Aliquots of substrate corresponding to 150 g drysolids were transferred to 500 ml blue cap glass flasks and placed in awater bath with stirring at the respective temperatures. Enzymes wereadded and pH readjusted if necessary (measured at incubationtemperature). Samples were taken periodically and analysed at HPLC fordetermination of the carbohydrate composition.

The glucose produced during saccharification are given in the tablebelow, the first three columns representing the saccharification withglucoamylase and acid α-amylase and pullulanase, the last three withglucoamylase alone. Numbers are % DP1 on DS.

0.24 AGU + 0.02 AFAU + Time 0.03 PUN 0.32 AGU (hours) 60° C. 65° C. 70°C. 60° C. 65° C. 70° C. 24 88.96 90.51 87.91 84.98 86.28 84.35 48 94.0394.28 91.90 88.86 89.51 86.98 72 95.08 94.75 93.12 90.18 90.42 87.99 9895.03 94.59 93.64 90.65 90.72 88.51

A glucose yield above 95% was obtained after 72 hours using an enzymedosage of 0.24 AGU/g DS which is corresponding to 0.03 mg/g DS. Thetypical dosage of A. niger AMG would be 0.18 AGU/g DS which iscorresponding to 0.09 mg/g DS to get a yield og 95-96% glucose. Asignificantly lower enzyme dosage on mg enzyme protein of TalaromycesAMG is therefore required in the saccharification process compared to A.niger AMG due to the high specific activity of T. emersonii AMG.

Example 12

Temperature Stability—T½ (half-life) of Recombinant Talaromycesemersonii AMG Expressed in Yeast

The thermal stability of recombinant Talaromyces emersonii glucoamylaseexpressed in yeast (purified using the method described in Example 9)was determined at 70° C., pH 4.5, 0.2 AGU/ml using the method describedabove in the “Material and Methods” section as “Thermal Stability I (T½(half-life) determination of AMG”.

FIG. 13 shows the result of the test. The T½ of the recombinantTalaromyces emersonii glucoamylase expressed in yeast was determined toabout 110 minutes at 70° C.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 34 <210> SEQ ID NO 1 <211>LENGTH: 25 <212> TYPE: PRT <213> ORGANISM: Talaromyces emersonii <220>FEATURE: <221> NAME/KEY: UNSURE <222> LOCATION: (0)...(0) <223> OTHERINFORMATION: Xaa at position 13 denotes a residue that could not beassigned <400> SEQUENCE: 1 Ala Asn Gly Ser Leu Asp Ser Phe Leu Ala ThrGlu Xaa Pro Ile Ala 1 5 10 15 Leu Gln Gly Val Leu Asn Asn Ile Gly 20 25<210> SEQ ID NO 2 <211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:Talaromyces emersonii <400> SEQUENCE: 2 Val Gln Thr Ile Ser Asn Pro SerGly Asp Leu Ser Thr Gly Gly Leu 1 5 10 15 Gly Glu Pro Lys 20 <210> SEQID NO 3 <211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM: Talaromycesemersonii <220> FEATURE: <221> NAME/KEY: UNSURE <222> LOCATION:(0)...(0) <223> OTHER INFORMATION: Xaa at positions 1 and 12 denotes aresidue that could not be assigned <400> SEQUENCE: 3 Xaa Asn Val Asn GluThr Ala Phe Thr Gly Pro Xaa Gly Arg Pro Gln 1 5 10 15 Arg Asp Gly ProAla Leu 20 <210> SEQ ID NO 4 <211> LENGTH: 35 <212> TYPE: PRT <213>ORGANISM: Talaromyces emersonii <400> SEQUENCE: 4 Asp Val Asn Ser IleLeu Gly Ser Ile His Thr Phe Asp Pro Ala Gly 1 5 10 15 Gly Cys Asp AspSer Thr Phe Gln Pro Cys Ser Ala Arg Ala Leu Ala 20 25 30 Asn His Lys 35<210> SEQ ID NO 5 <211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:Talaromyces emersonii <220> FEATURE: <221> NAME/KEY: UNSURE <222>LOCATION: (0)...(0) <223> OTHER INFORMATION: Xaa at position 2 denotes aresidue that could not be assigned <400> SEQUENCE: 5 Thr Xaa Ala Ala AlaGlu Gln Leu Tyr Asp Ala Ile Tyr Gln Trp Lys 1 5 10 15 <210> SEQ ID NO 6<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: Talaromyces emersonii<400> SEQUENCE: 6 Ala Gln Thr Asp Gly Thr Ile Val Trp Glu Asp Asp ProAsn Arg Ser 1 5 10 15 Tyr Thr Val Pro Ala Tyr Cys Gly Gln Thr Thr AlaIle Leu Asp Asp 20 25 30 Ser Trp Gln 35 <210> SEQ ID NO 7 <211> LENGTH:591 <212> TYPE: PRT <213> ORGANISM: Talaromyces emersonii <400>SEQUENCE: 7 Ala Thr Gly Ser Leu Asp Ser Phe Leu Ala Thr Glu Thr Pro IleAla 1 5 10 15 Leu Gln Gly Val Leu Asn Asn Ile Gly Pro Asn Gly Ala AspVal Ala 20 25 30 Gly Ala Ser Ala Gly Ile Val Val Ala Ser Pro Ser Arg SerAsp Pro 35 40 45 Asn Tyr Phe Tyr Ser Trp Thr Arg Asp Ala Ala Leu Thr AlaLys Tyr 50 55 60 Leu Val Asp Ala Phe Asn Arg Gly Asn Lys Asp Leu Glu GlnThr Ile 65 70 75 80 Gln Gln Tyr Ile Ser Ala Gln Ala Lys Val Gln Thr IleSer Asn Pro 85 90 95 Ser Gly Asp Leu Ser Thr Gly Gly Leu Gly Glu Pro LysPhe Asn Val 100 105 110 Asn Glu Thr Ala Phe Thr Gly Pro Trp Gly Arg ProGln Arg Asp Gly 115 120 125 Pro Ala Leu Arg Ala Thr Ala Leu Ile Ala TyrAla Asn Tyr Leu Ile 130 135 140 Asp Asn Gly Glu Ala Ser Thr Ala Asp GluIle Ile Trp Pro Ile Val 145 150 155 160 Gln Asn Asp Leu Ser Tyr Ile ThrGln Tyr Trp Asn Ser Ser Thr Phe 165 170 175 Asp Leu Trp Glu Glu Val GluGly Ser Ser Phe Phe Thr Thr Ala Val 180 185 190 Gln His Arg Ala Leu ValGlu Gly Asn Ala Leu Ala Thr Arg Leu Asn 195 200 205 His Thr Cys Ser AsnCys Val Ser Gln Ala Pro Gln Val Leu Cys Phe 210 215 220 Leu Gln Ser TyrTrp Thr Gly Ser Tyr Val Leu Ala Asn Phe Gly Gly 225 230 235 240 Ser GlyArg Ser Gly Lys Asp Val Asn Ser Ile Leu Gly Ser Ile His 245 250 255 ThrPhe Asp Pro Ala Gly Gly Cys Asp Asp Ser Thr Phe Gln Pro Cys 260 265 270Ser Ala Arg Ala Leu Ala Asn His Lys Val Val Thr Asp Ser Phe Arg 275 280285 Ser Ile Tyr Ala Ile Asn Ser Gly Ile Ala Glu Gly Ser Ala Val Ala 290295 300 Val Gly Arg Tyr Pro Glu Asp Val Tyr Gln Gly Gly Asn Pro Trp Tyr305 310 315 320 Leu Ala Thr Ala Ala Ala Ala Glu Gln Leu Tyr Asp Ala IleTyr Gln 325 330 335 Trp Lys Lys Ile Gly Ser Ile Ser Ile Thr Asp Val SerLeu Pro Phe 340 345 350 Phe Gln Asp Ile Tyr Pro Ser Ala Ala Val Gly ThrTyr Asn Ser Gly 355 360 365 Ser Thr Thr Phe Asn Asp Ile Ile Ser Ala ValGln Thr Tyr Gly Asp 370 375 380 Gly Tyr Leu Ser Ile Val Glu Lys Tyr ThrPro Ser Asp Gly Ser Leu 385 390 395 400 Thr Glu Gln Phe Ser Arg Thr AspGly Thr Pro Leu Ser Ala Ser Ala 405 410 415 Leu Thr Trp Ser Tyr Ala SerLeu Leu Thr Ala Ser Ala Arg Arg Gln 420 425 430 Ser Val Val Pro Ala SerTrp Gly Glu Ser Ser Ala Ser Ser Val Leu 435 440 445 Ala Val Cys Ser AlaThr Ser Ala Thr Gly Pro Tyr Ser Thr Ala Thr 450 455 460 Asn Thr Val TrpPro Ser Ser Gly Ser Gly Ser Ser Thr Thr Thr Ser 465 470 475 480 Ser AlaPro Cys Thr Thr Pro Thr Ser Val Ala Val Thr Phe Asp Glu 485 490 495 IleVal Ser Thr Ser Tyr Gly Glu Thr Ile Tyr Leu Ala Gly Ser Ile 500 505 510Pro Glu Leu Gly Asn Trp Ser Thr Ala Ser Ala Ile Pro Leu Arg Ala 515 520525 Asp Ala Tyr Thr Asn Ser Asn Pro Leu Trp Tyr Val Thr Val Asn Leu 530535 540 Pro Pro Gly Thr Ser Phe Glu Tyr Lys Phe Phe Lys Asn Gln Thr Asp545 550 555 560 Gly Thr Ile Val Trp Glu Asp Asp Pro Asn Arg Ser Tyr ThrVal Pro 565 570 575 Ala Tyr Cys Gly Gln Thr Thr Ala Ile Leu Asp Asp SerTrp Gln 580 585 590 <210> SEQ ID NO 8 <211> LENGTH: 1605 <212> TYPE: DNA<213> ORGANISM: Aspergillus niger <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (1)...(1602) <221> NAME/KEY: sig_peptide <222> LOCATION:(1)...(72) <221> NAME/KEY: mat_peptide <222> LOCATION: (73)...(1602)<400> SEQUENCE: 8 atg tcg ttc cga tct cta ctc gcc ctg agc ggc ctc gtctgc aca ggg 48 Met Ser Phe Arg Ser Leu Leu Ala Leu Ser Gly Leu Val CysThr Gly -20 -15 -10 ttg gca aat gtg att tcc aag cgc gcg acc ttg gat tcatgg ttg agc 96 Leu Ala Asn Val Ile Ser Lys Arg Ala Thr Leu Asp Ser TrpLeu Ser -5 1 5 aac gaa gcg acc gtg gct cgt act gcc atc ctg aat aac atcggg gcg 144 Asn Glu Ala Thr Val Ala Arg Thr Ala Ile Leu Asn Asn Ile GlyAla 10 15 20 gac ggt gct tgg gtg tcg ggc gcg gac tct ggc att gtc gtt gctagt 192 Asp Gly Ala Trp Val Ser Gly Ala Asp Ser Gly Ile Val Val Ala Ser25 30 35 40 ccc agc acg gat aac ccg gac tac ttc tac acc tgg act cgc gactct 240 Pro Ser Thr Asp Asn Pro Asp Tyr Phe Tyr Thr Trp Thr Arg Asp Ser45 50 55 ggt ctc gtc ctc aag acc ctc gtc gat ctc ttc cga aat gga gat acc288 Gly Leu Val Leu Lys Thr Leu Val Asp Leu Phe Arg Asn Gly Asp Thr 6065 70 agt ctc ctc tcc acc att gag aac tac atc tcc gcc cag gca att gtc336 Ser Leu Leu Ser Thr Ile Glu Asn Tyr Ile Ser Ala Gln Ala Ile Val 7580 85 cag ggt atc agt aac ccc tct ggt gat ctg tcc agc ggc gct ggt ctc384 Gln Gly Ile Ser Asn Pro Ser Gly Asp Leu Ser Ser Gly Ala Gly Leu 9095 100 ggt gaa ccc aag ttc aat gtc gat gag act gcc tac act ggt tct tgg432 Gly Glu Pro Lys Phe Asn Val Asp Glu Thr Ala Tyr Thr Gly Ser Trp 105110 115 120 gga cgg ccg cag cga gat ggt ccg gct ctg aga gca act gct atgatc 480 Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala Thr Ala Met Ile125 130 135 ggc ttc ggg cag tgg ctg ctt gac aat ggc tac acc agc acc gcaacg 528 Gly Phe Gly Gln Trp Leu Leu Asp Asn Gly Tyr Thr Ser Thr Ala Thr140 145 150 gac att gtt tgg ccc ctc gtt agg aac gac ctg tcg tat gtg gctcaa 576 Asp Ile Val Trp Pro Leu Val Arg Asn Asp Leu Ser Tyr Val Ala Gln155 160 165 tac tgg aac cag aca gga tat gat ctc tgg gaa gaa gtc aat ggctcg 624 Tyr Trp Asn Gln Thr Gly Tyr Asp Leu Trp Glu Glu Val Asn Gly Ser170 175 180 tct ttc ttt acg att gct gtg caa cac cgc gcc ctt gtc gaa ggtagt 672 Ser Phe Phe Thr Ile Ala Val Gln His Arg Ala Leu Val Glu Gly Ser185 190 195 200 gcc ttc gcg acg gcc gtc ggc tcg tcc tgc tcc tgg tgt gattct cag 720 Ala Phe Ala Thr Ala Val Gly Ser Ser Cys Ser Trp Cys Asp SerGln 205 210 215 gca ccc gaa att ctc tgc tac ctg cag tcc ttc tgg acc ggcagc ttc 768 Ala Pro Glu Ile Leu Cys Tyr Leu Gln Ser Phe Trp Thr Gly SerPhe 220 225 230 att ctg gcc aac ttc gat agc agc cgt tcc ggc aag gac gcaaac acc 816 Ile Leu Ala Asn Phe Asp Ser Ser Arg Ser Gly Lys Asp Ala AsnThr 235 240 245 ctc ctg gga agc atc cac acc ttt gat cct gag gcc gca tgcgac gac 864 Leu Leu Gly Ser Ile His Thr Phe Asp Pro Glu Ala Ala Cys AspAsp 250 255 260 tcc acc ttc cag ccc tgc tcc ccg cgc gcg ctc gcc aac cacaag gag 912 Ser Thr Phe Gln Pro Cys Ser Pro Arg Ala Leu Ala Asn His LysGlu 265 270 275 280 gtt gta gac tct ttc cgc tca atc tat acc ctc aac gatggt ctc agt 960 Val Val Asp Ser Phe Arg Ser Ile Tyr Thr Leu Asn Asp GlyLeu Ser 285 290 295 gac agc gag gct gtt gcg gtg ggt cgg tac cct gag gacacg tac tac 1008 Asp Ser Glu Ala Val Ala Val Gly Arg Tyr Pro Glu Asp ThrTyr Tyr 300 305 310 aac ggc aac ccg tgg ttc ctg tgc acc ttg gct gcc gcagag cag ttg 1056 Asn Gly Asn Pro Trp Phe Leu Cys Thr Leu Ala Ala Ala GluGln Leu 315 320 325 tac gat gct cta tac cag tgg gac aag cag ggg tcg ttggag gtc aca 1104 Tyr Asp Ala Leu Tyr Gln Trp Asp Lys Gln Gly Ser Leu GluVal Thr 330 335 340 gat gtg tcg ctg gac ttc ttc aag gca ctg tac agc gatgct gct act 1152 Asp Val Ser Leu Asp Phe Phe Lys Ala Leu Tyr Ser Asp AlaAla Thr 345 350 355 360 ggc acc tac tct tcg tcc agt tcg act tat agt agcatt gta gat gcc 1200 Gly Thr Tyr Ser Ser Ser Ser Ser Thr Tyr Ser Ser IleVal Asp Ala 365 370 375 gtg aag act ttc gcc gat ggc ttc gtc tct att gtggaa act cac gcc 1248 Val Lys Thr Phe Ala Asp Gly Phe Val Ser Ile Val GluThr His Ala 380 385 390 gca agc aac ggc tcc atg tcc gag caa tac gac aagtct gat ggc gag 1296 Ala Ser Asn Gly Ser Met Ser Glu Gln Tyr Asp Lys SerAsp Gly Glu 395 400 405 cag ctt tcc gct cgc gac ctg acc tgg tct tat gctgct ctg ctg acc 1344 Gln Leu Ser Ala Arg Asp Leu Thr Trp Ser Tyr Ala AlaLeu Leu Thr 410 415 420 gcc aac aac cgt cgt aac tcc gtc gtg cct gct tcttgg ggc gag acc 1392 Ala Asn Asn Arg Arg Asn Ser Val Val Pro Ala Ser TrpGly Glu Thr 425 430 435 440 tct gcc agc agc gtg ccc ggc acc tgt gcg gccaca tct gcc att ggt 1440 Ser Ala Ser Ser Val Pro Gly Thr Cys Ala Ala ThrSer Ala Ile Gly 445 450 455 acc tac agc agt gtg act gtc acc tcg tgg ccgagt atc gtg gct act 1488 Thr Tyr Ser Ser Val Thr Val Thr Ser Trp Pro SerIle Val Ala Thr 460 465 470 ggc ggc acc act acg acg gct acc ccc act ggatcc ggc agc gtg acc 1536 Gly Gly Thr Thr Thr Thr Ala Thr Pro Thr Gly SerGly Ser Val Thr 475 480 485 tcg acc agc aag acc acc gcg act gct agc aagacc agc acc acg acc 1584 Ser Thr Ser Lys Thr Thr Ala Thr Ala Ser Lys ThrSer Thr Thr Thr 490 495 500 cgc tct ggt atg tca ctg tga 1605 Arg Ser GlyMet Ser Leu 505 510 <210> SEQ ID NO 9 <211> LENGTH: 534 <212> TYPE: PRT<213> ORGANISM: Aspergillus niger <220> FEATURE: <221> NAME/KEY: SIGNAL<222> LOCATION: (1)...(24) <400> SEQUENCE: 9 Met Ser Phe Arg Ser Leu LeuAla Leu Ser Gly Leu Val Cys Thr Gly -20 -15 -10 Leu Ala Asn Val Ile SerLys Arg Ala Thr Leu Asp Ser Trp Leu Ser -5 1 5 Asn Glu Ala Thr Val AlaArg Thr Ala Ile Leu Asn Asn Ile Gly Ala 10 15 20 Asp Gly Ala Trp Val SerGly Ala Asp Ser Gly Ile Val Val Ala Ser 25 30 35 40 Pro Ser Thr Asp AsnPro Asp Tyr Phe Tyr Thr Trp Thr Arg Asp Ser 45 50 55 Gly Leu Val Leu LysThr Leu Val Asp Leu Phe Arg Asn Gly Asp Thr 60 65 70 Ser Leu Leu Ser ThrIle Glu Asn Tyr Ile Ser Ala Gln Ala Ile Val 75 80 85 Gln Gly Ile Ser AsnPro Ser Gly Asp Leu Ser Ser Gly Ala Gly Leu 90 95 100 Gly Glu Pro LysPhe Asn Val Asp Glu Thr Ala Tyr Thr Gly Ser Trp 105 110 115 120 Gly ArgPro Gln Arg Asp Gly Pro Ala Leu Arg Ala Thr Ala Met Ile 125 130 135 GlyPhe Gly Gln Trp Leu Leu Asp Asn Gly Tyr Thr Ser Thr Ala Thr 140 145 150Asp Ile Val Trp Pro Leu Val Arg Asn Asp Leu Ser Tyr Val Ala Gln 155 160165 Tyr Trp Asn Gln Thr Gly Tyr Asp Leu Trp Glu Glu Val Asn Gly Ser 170175 180 Ser Phe Phe Thr Ile Ala Val Gln His Arg Ala Leu Val Glu Gly Ser185 190 195 200 Ala Phe Ala Thr Ala Val Gly Ser Ser Cys Ser Trp Cys AspSer Gln 205 210 215 Ala Pro Glu Ile Leu Cys Tyr Leu Gln Ser Phe Trp ThrGly Ser Phe 220 225 230 Ile Leu Ala Asn Phe Asp Ser Ser Arg Ser Gly LysAsp Ala Asn Thr 235 240 245 Leu Leu Gly Ser Ile His Thr Phe Asp Pro GluAla Ala Cys Asp Asp 250 255 260 Ser Thr Phe Gln Pro Cys Ser Pro Arg AlaLeu Ala Asn His Lys Glu 265 270 275 280 Val Val Asp Ser Phe Arg Ser IleTyr Thr Leu Asn Asp Gly Leu Ser 285 290 295 Asp Ser Glu Ala Val Ala ValGly Arg Tyr Pro Glu Asp Thr Tyr Tyr 300 305 310 Asn Gly Asn Pro Trp PheLeu Cys Thr Leu Ala Ala Ala Glu Gln Leu 315 320 325 Tyr Asp Ala Leu TyrGln Trp Asp Lys Gln Gly Ser Leu Glu Val Thr 330 335 340 Asp Val Ser LeuAsp Phe Phe Lys Ala Leu Tyr Ser Asp Ala Ala Thr 345 350 355 360 Gly ThrTyr Ser Ser Ser Ser Ser Thr Tyr Ser Ser Ile Val Asp Ala 365 370 375 ValLys Thr Phe Ala Asp Gly Phe Val Ser Ile Val Glu Thr His Ala 380 385 390Ala Ser Asn Gly Ser Met Ser Glu Gln Tyr Asp Lys Ser Asp Gly Glu 395 400405 Gln Leu Ser Ala Arg Asp Leu Thr Trp Ser Tyr Ala Ala Leu Leu Thr 410415 420 Ala Asn Asn Arg Arg Asn Ser Val Val Pro Ala Ser Trp Gly Glu Thr425 430 435 440 Ser Ala Ser Ser Val Pro Gly Thr Cys Ala Ala Thr Ser AlaIle Gly 445 450 455 Thr Tyr Ser Ser Val Thr Val Thr Ser Trp Pro Ser IleVal Ala Thr 460 465 470 Gly Gly Thr Thr Thr Thr Ala Thr Pro Thr Gly SerGly Ser Val Thr 475 480 485 Ser Thr Ser Lys Thr Thr Ala Thr Ala Ser LysThr Ser Thr Thr Thr 490 495 500 Arg Ser Gly Met Ser Leu 505 510 <210>SEQ ID NO 10 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Primer 102434 N= A, G,C or T <400> SEQUENCE: 10 gtnttraaya ayathgg 17 <210> SEQ ID NO 11 <211>LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Primer 102435 N = A, G, C, or T <400>SEQUENCE: 11 gtnctnaaya ayathgg 17 <210> SEQ ID NO 12 <211> LENGTH: 17<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Primer 117360 N= A, G, C or T <400> SEQUENCE: 12ctrganaccc tyctyca 17 <210> SEQ ID NO 13 <211> LENGTH: 17 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Primer 117361 <400> SEQUENCE: 13 ctraayaccc tyctyca 17<210> SEQ ID NO 14 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer127420 N= A, G, C or T <400> SEQUENCE: 14 accctyctrc trggntt 17 <210>SEQ ID NO 15 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Primer 123036 <400>SEQUENCE: 15 gtgagcccaa gttcaatgtg 20 <210> SEQ ID NO 16 <211> LENGTH:21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer 1 <400> SEQUENCE: 16 agaaatcgggtatcctttca g 21 <210> SEQ ID NO 17 <211> LENGTH: 105 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Primer 2 <400> SEQUENCE: 17 gctcctcatg gtggatccccagttgtgtat atagaggatt gaggaaggaa gagaagtgtg 60 gatagaggta aattgagttggaaactccaa gcatggcatc cttgc 105 <210> SEQ ID NO 18 <211> LENGTH: 30<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Primer 139746 <400> SEQUENCE: 18 gacagatctccaccatggcg tccctcgttg 30 <210> SEQ ID NO 19 <211> LENGTH: 27 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Primer 3 <400> SEQUENCE: 19 gacctcgagt cactgccaac tatcgtc27 <210> SEQ ID NO 20 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer 4<400> SEQUENCE: 20 ccctcaccag gggaatgctg cagttgatg 29 <210> SEQ ID NO 21<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Primer 950847 <400> SEQUENCE: 21cgccattctc ggcgactt 18 <210> SEQ ID NO 22 <211> LENGTH: 18 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Primer 951216 <400> SEQUENCE: 22 cgccgcggta ttctgcag 18<210> SEQ ID NO 23 <211> LENGTH: 50 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer Tal 1<400> SEQUENCE: 23 caatataaac gacggtaccc gggagatctc caccatggcgtccctcgttg 50 <210> SEQ ID NO 24 <211> LENGTH: 44 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Tal 4 <400> SEQUENCE: 24 ctaattacat catgcggccc tctagatcac tgccaactatcgtc 44 <210> SEQ ID NO 25 <211> LENGTH: 21 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Primer Tal 5 <400> SEQUENCE: 25 aatttgggtc gctcctgctc g 21 <210> SEQ IDNO 26 <211> LENGTH: 40 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Tal 6 <400> SEQUENCE:26 cgagcaggag cgacccaaat tatttctact cctggacacg 40 <210> SEQ ID NO 27<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Tal 7 <400> SEQUENCE: 27gatgagatag ttcgcatacg 20 <210> SEQ ID NO 28 <211> LENGTH: 43 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Tal 8 <400> SEQUENCE: 28 cgtatgcgaa ctatctcatc gacaacggcgaggcttcgac tgc 43 <210> SEQ ID NO 29 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Tal 9 <400> SEQUENCE: 29 cgaaggtgga tgagttccag 20 <210> SEQID NO 30 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Tal 10 <400> SEQUENCE:30 ctggaactca tccaccttcg acctctggga agaagtagaa gg 42 <210> SEQ ID NO 31<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Tal 11 <400> SEQUENCE: 31gacaatactc agatatccat c 21 <210> SEQ ID NO 32 <211> LENGTH: 43 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Tal 12 <400> SEQUENCE: 32 gatggatatc tgagtattgt cgagaaatatactccctcag acg 43 <210> SEQ ID NO 33 <211> LENGTH: 2748 <212> TYPE: DNA<213> ORGANISM: Talaromyces emersonii <400> SEQUENCE: 33 acgagatgtgtatatactgt gaaccaaact agatgatgtc agttatgctg gtctgagaac 60 tcatagaagcccttgaaaat accccaagct agcactccaa ccctaactct gttgctctac 120 tagatcaagacgagtactct gattgagctg caggcttgga atatatgatt agcagaaaaa 180 gggttaaaacttgtatgaca atcagtttgt cagtactccg tagtgatgcc atgtctatag 240 agtcgacactaaggcagcat gtgaatgagt cggaaatgac aggaagcaga ttccttaaca 300 gtcatgttctccgtgcctgc atccccacgt cacctgcaaa gatgcgacgc tactccacac 360 cggcgccttgatgtctgctg ttcctggcct agtggagccc catgcgctgc tagctcgtgg 420 tcttcgaataaatcagaata aaaaacggag taattaattg cgcccgcaac aaactaagca 480 atgtaactcaatgccaagct tccgctgatg ctcttgacat ctccgtagtg gcttctttcg 540 taatttcagacgtatatata gtagtaatgc ccagcaggcc gggataatga tggggatttc 600 tgaactctcagcttccgtac gctgaacagt ttgcttgcgt tgtcaaccat ggcgtccctc 660 gttgctggcgctctctgcat cctgggcctg acgcctgctg catttgcacg agcgcccgtt 720 gcagcgcgagccaccggttc cctggactcc tttctcgcaa ccgaaactcc aattgccctc 780 caaggcgtgctgaacaacat cgggcccaat ggtgctgatg tggcaggagc aagcgccggc 840 attgtggttgccagtccgag caggagcgac ccaaattgta ggttctttcc caccagaaat 900 tacttatttaaatcagccct ctgacaggtt gaagatttct actcctggac acgtgacgca 960 gcgctcacggccaaatacct cgtcgacgcc ttcatcgcgg gcaacaagga cctagagcag 1020 accatccagcagtacatcag cgcgcaggcg aaggtgcaaa ctatctccaa tccgtccgga 1080 gatttatccaccggtggctt aggtgagccc aagttcaatg tgaatgagac ggcttttacc 1140 gggccctggggtcgtccaca gagggacgga ccagcgttga gagcgacggc cctcattgcg 1200 tatgcgaactatctcatcgt aagcttctgc tcgctgccct tctctctgct cgtatgctaa 1260 gtagtcctgtcaggacaacg gcgaggcttc gactgccgat gagatcatct ggccgattgt 1320 ccagaatgatctgtcctaca tcacccaata ctggaactca tccaccttcg gtaggcaaat 1380 gaatattcccgacacagcgt ggtactaatt tgattcagac ctctgggaag aagtagaagg 1440 atcctcattcttcacaaccg ccgtgcaaca ccgcgccctg gtcgaaggca atgcactggc 1500 aacaaggctgaaccacacgt gctccaactg cgtctctcag gcccctcagg tcctgtgttt 1560 cctgcagtcatactggaccg gatcgtatgt tctggccaac tttggtggca gcggtcgttc 1620 cggcaaggacgtgaattcga ttctgggcag catccacacc tttgatcccg ccggaggctg 1680 tgacgactcgaccttccagc cgtgttcggc ccgtgccttg gcaaatcaca aggtggtcac 1740 cgactcgttccggagtatct atgcgatcaa ctcaggcatc gcagagggat ctgccgtggc 1800 agtcggccgctaccctgagg atgtctacca gggcgggaac ccctggtacc tggccacagc 1860 agcggctgcagagcagcttt acgacgccat ctaccagtgg aagaagatcg gctcgataag 1920 tatcacggacgttagtctgc catttttcca ggatatctac ccttctgccg cggtgggcac 1980 ctataactctggctccacga ctttcaacga catcatctcg gccgtccaga cgtatggtga 2040 tggatatctgagtattgtcg tacgttttgc cttagattct caggtgtaaa gaaaaaaatg 2100 gaactaactcagttctagga gaaatatact ccctcagacg gctctcttac cgaacaattc 2160 tcccgtacagacggcactcc gctttctgcc tctgccctga cttggtcgta cgcttctctc 2220 ctaaccgcttcggcccgcag acagtccgtc gtccctgctt cctggggcga aagctccgca 2280 agcagcgtccctgccgtctg ctctgccacc tctgccacgg gcccatacag cacggctacc 2340 aacaccgtctggccaagctc tggctctggc agctcaacaa ccaccagtag cgccccatgc 2400 accactcctacctctgtggc tgtgaccttc gacgaaatcg tcagcaccag ttacggggag 2460 acaatctacctggccggctc gatccccgag ctgggcaact ggtccacggc cagcgcgatc 2520 cccctccgcgcggatgctta caccaacagc aacccgctct ggtacgtgac cgtcaatctg 2580 ccccctggcaccagcttcga gtacaagttc ttcaagaacc agacggacgg gaccatcgtc 2640 tgggaagacgacccgaaccg gtcgtacacg gtcccagcgt actgtgggca gactaccgcc 2700 attcttgacgatagttggca gtgagataac atccaccctt ctgtttta 2748 <210> SEQ ID NO 34 <211>LENGTH: 618 <212> TYPE: PRT <213> ORGANISM: Talaromyces emersonii <400>SEQUENCE: 34 Met Ala Ser Leu Val Ala Gly Ala Leu Cys Ile Leu Gly Leu ThrPro 1 5 10 15 Ala Ala Phe Ala Arg Ala Pro Val Ala Ala Arg Ala Thr GlySer Leu 20 25 30 Asp Ser Phe Leu Ala Thr Glu Thr Pro Ile Ala Leu Gln GlyVal Leu 35 40 45 Asn Asn Ile Gly Pro Asn Gly Ala Asp Val Ala Gly Ala SerAla Gly 50 55 60 Ile Val Val Ala Ser Pro Ser Arg Ser Asp Pro Asn Tyr PheTyr Ser 65 70 75 80 Trp Thr Arg Asp Ala Ala Leu Thr Ala Lys Tyr Leu ValAsp Ala Phe 85 90 95 Ile Ala Gly Asn Lys Asp Leu Glu Gln Thr Ile Gln GlnTyr Ile Ser 100 105 110 Ala Gln Ala Lys Val Gln Thr Ile Ser Asn Pro SerGly Asp Leu Ser 115 120 125 Thr Gly Gly Leu Gly Glu Pro Lys Phe Asn ValAsn Glu Thr Ala Phe 130 135 140 Thr Gly Pro Trp Gly Arg Pro Gln Arg AspGly Pro Ala Leu Arg Ala 145 150 155 160 Thr Ala Leu Ile Ala Tyr Ala AsnTyr Leu Ile Asp Asn Gly Glu Ala 165 170 175 Ser Thr Ala Asp Glu Ile IleTrp Pro Ile Val Gln Asn Asp Leu Ser 180 185 190 Tyr Ile Thr Gln Tyr TrpAsn Ser Ser Thr Phe Asp Leu Trp Glu Glu 195 200 205 Val Glu Gly Ser SerPhe Phe Thr Thr Ala Val Gln His Arg Ala Leu 210 215 220 Val Glu Gly AsnAla Leu Ala Thr Arg Leu Asn His Thr Cys Ser Asn 225 230 235 240 Cys ValSer Gln Ala Pro Gln Val Leu Cys Phe Leu Gln Ser Tyr Trp 245 250 255 ThrGly Ser Tyr Val Leu Ala Asn Phe Gly Gly Ser Gly Arg Ser Gly 260 265 270Lys Asp Val Asn Ser Ile Leu Gly Ser Ile His Thr Phe Asp Pro Ala 275 280285 Gly Gly Cys Asp Asp Ser Thr Phe Gln Pro Cys Ser Ala Arg Ala Leu 290295 300 Ala Asn His Lys Val Val Thr Asp Ser Phe Arg Ser Ile Tyr Ala Ile305 310 315 320 Asn Ser Gly Ile Ala Glu Gly Ser Ala Val Ala Val Gly ArgTyr Pro 325 330 335 Glu Asp Val Tyr Gln Gly Gly Asn Pro Trp Tyr Leu AlaThr Ala Ala 340 345 350 Ala Ala Glu Gln Leu Tyr Asp Ala Ile Tyr Gln TrpLys Lys Ile Gly 355 360 365 Ser Ile Ser Ile Thr Asp Val Ser Leu Pro PhePhe Gln Asp Ile Tyr 370 375 380 Pro Ser Ala Ala Val Gly Thr Tyr Asn SerGly Ser Thr Thr Phe Asn 385 390 395 400 Asp Ile Ile Ser Ala Val Gln ThrTyr Gly Asp Gly Tyr Leu Ser Ile 405 410 415 Val Glu Lys Tyr Thr Pro SerAsp Gly Ser Leu Thr Glu Gln Phe Ser 420 425 430 Arg Thr Asp Gly Thr ProLeu Ser Ala Ser Ala Leu Thr Trp Ser Tyr 435 440 445 Ala Ser Leu Leu ThrAla Ser Ala Arg Arg Gln Ser Val Val Pro Ala 450 455 460 Ser Trp Gly GluSer Ser Ala Ser Ser Val Pro Ala Val Cys Ser Ala 465 470 475 480 Thr SerAla Thr Gly Pro Tyr Ser Thr Ala Thr Asn Thr Val Trp Pro 485 490 495 SerSer Gly Ser Gly Ser Ser Thr Thr Thr Ser Ser Ala Pro Cys Thr 500 505 510Thr Pro Thr Ser Val Ala Val Thr Phe Asp Glu Ile Val Ser Thr Ser 515 520525 Tyr Gly Glu Thr Ile Tyr Leu Ala Gly Ser Ile Pro Glu Leu Gly Asn 530535 540 Trp Ser Thr Ala Ser Ala Ile Pro Leu Arg Ala Asp Ala Tyr Thr Asn545 550 555 560 Ser Asn Pro Leu Trp Tyr Val Thr Val Asn Leu Pro Pro GlyThr Ser 565 570 575 Phe Glu Tyr Lys Phe Phe Lys Asn Gln Thr Asp Gly ThrIle Val Trp 580 585 590 Glu Asp Asp Pro Asn Arg Ser Tyr Thr Val Pro AlaTyr Cys Gly Gln 595 600 605 Thr Thr Ala Ile Leu Asp Asp Ser Trp Gln 610615

What is claimed is:
 1. An isolated enzyme with glucoamylase activity,wherein the enzyme (a) is derived from Talaromyces and has a T_(½)(half-life) of at least 100 minutes in 50 mM NaOAc, 0.2 NovoAmyloglucosidase Unit (AGU)/ml, pH 4.5, at 70° C. or (b) has an aminoacid sequence that has at least 80% identity with the glucoamylase ofSEQ ID NO:7.
 2. The enzyme of claim 1, wherein the enzyme is derivedfrom Talaromyces and has a T_(½) (half-life) of at least 100 minutes in50 mM NaOAc, 0.2 Novo Amyloglucosidase Unit (AGU)/ml, pH 4.5, at 70° C.3. The enzyme of claim 1, wherein the enzyme has an amino acid sequencethat is at least 80% identical with the glucoamylase of SEQ ID NO:7. 4.The enzyme of claim 3, wherein the enzyme has an amino acid sequencethat is at least 90% identical with the glucoamylase of SEQ ID NO:7. 5.The enzyme of claim 4, wherein the enzyme has an amino acid sequencethat is at least 95% identical with the glucoamylase of SEQ ID NO:7. 6.The enzyme of claim 5, wherein the enzyme has an amino acid sequencethat is at least 97% identical with the glucoamylase of SEQ ID NO:7. 7.The enzyme of claim 6, wherein the enzyme has an amino acid sequencethat is at least 99% identical with the glucoamylase of SEQ ID NO:7. 8.The enzyme of claim 1 which has a molecular weight of about 70 kDadetermined by SDS-PAGE.
 9. The enzyme of claim 1 which has anisoelectric point below 3.5 determined by isoelectric focusing.
 10. Theenzyme of claim 1 which is derived from a fungal organism.
 11. Theenzyme of claim 10 which is derived from a filamentous fungus.
 12. Theenzyme of claim 11, wherein the filamentous fungus is a Talaromycesstrain.
 13. The enzyme of claim 12, wherein the filamentous fungus is aTalaromyces emersonii strain.
 14. The enzyme of claim 13, wherein thefilamentous fungus is Talaromyces emersonii CBS 793.97.
 15. An isolatedenzyme having an amino acid sequence comprising amino acids 28-618 ofSEQ ID NO:34.
 16. The enzyme of claim 15 having an amino acid sequenceconsisting of amino acids 28-618 of SEQ ID NO:34.
 17. The enzyme ofclaim 16 having an amino acid sequence comprising amino acids 1-618 ofSEQ ID NO:34.
 18. An isolated enzyme having an amino acid sequencecomprising amino acids 28-618 of SEQ ID NO:34 and the substitution ofAsp at position 145 with Asn.
 19. A process for converting starch orpartially hydrolyzed starch into a syrup containing dextrose, comprisingsaccharifying a starch hydrolyzate in the presence of an enzyme of claim1.
 20. The process of claim 19, wherein the dosage of the enzyme is inthe range from 0.05 to 0.5 AGU per gram of dry solids.
 21. The processof claim 19, wherein the starch hydrolyzate has at least 30 percent byweight of dry solids.
 22. The process of claim 19, wherein thesaccharification is performed in the presence of a debranching enzymeselected from the group of pullulanase and isoamylase.
 23. The processof claim 19, wherein the saccharification is performed at a pH of about3 to 5.5, a temperature of 60-80° C., and for 24 to 72 hours.
 24. Amethod of saccharifying a liquefied starch solution, comprising applyingan enzyme of claim 1 to the liquefied starch solution.